Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs
Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs
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DOI：
10.1007/s11033-020-05267-z
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发表时间：
2020-02-03
影响因子：
2.8
通讯作者：
Miller, Heather B.
中科院分区：
文献类型：
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作者：
Hulver, Molly J.;Trautman, Julia P.;Miller, Heather B.
人类免疫缺陷病毒1型（HIV-1）的传播需要许多人类辅助因子。多个研究小组已经证明Tat特异性因子1（Tat-SF 1）是一种HIV-1依赖因子。这种蛋白质的缺失降低了HIV-1的感染性，但是，它不影响病毒RNA的总体水平。相反，Tat-SF 1调节每个RNA大小类别的相对水平。这将与病毒RNA的剪接、转运和/或稳定性中的作用一致。我们假设，如果Tat-SF 1发挥任何这些作用，那么我们应该检测蛋白质与RNA基因组的结合。此外，敲低Tat-SF 1应导致改变的RNA稳定性和/或在人细胞中的定位。HIV-1基因组片段被用作电泳迁移率变动分析和荧光相关光谱实验中的RNA探针。我们的研究结果表明，Tat-SF 1可以在体外与TAR RNA形成复合物，不依赖于达特。该因子与HIV-1基因组5'端的至少一个额外位置相互作用。达特似乎促进了这种复合物的形成。为了分析HIV-1 RNA定位，还用前病毒克隆转染Tat-SF 1敲低的HeLa细胞。纯化来自细胞核和细胞质级分的RNA，然后进行RT-qPCR分析。Tat-SF 1可能在TAR和潜在的其他位置结合HIV-1 RNA基因组，并选择性地转运HIV-1 RNA，促进未剪接RNA的核输出，同时将单剪接RNA保留在核中。这是HIV-1依赖因子的一个新作用。
Human immunodeficiency virus type 1 (HIV-1) propagation requires many human cofactors. Multiple groups have demonstrated that Tat-specific factor 1 (Tat-SF1) is an HIV-1 dependency factor. Depletion of this protein lowers HIV-1 infectivity, however, it does not affect the overall levels of viral RNA. Rather, Tat-SF1 regulates the relative levels of each RNA size class. This would be consistent with roles in splicing, transport, and/or stability of viral RNAs. We hypothesized that if Tat-SF1 plays any of these roles, then we should detect binding of the protein to the RNA genome. Furthermore, knocking down Tat-SF1 should result in altered RNA stability and/or localization in human cells. Fragments of the HIV-1 genome were used as RNA probes in electrophoretic mobility shift assays and fluorescence correlation spectroscopy experiments. Our results show that Tat-SF1 can form a complex with TAR RNA in vitro, independent of Tat. This factor interacts with at least one additional location in the 5' end of the HIV-1 genome. Tat seems to enhance the formation of this complex. To analyze HIV-1 RNA localization, HeLa cells with Tat-SF1 knocked down were also transfected with a proviral clone. RNA from nuclear and cytoplasmic fractions was purified, followed by RT-qPCR analysis. Tat-SF1 likely binds the HIV-1 RNA genome at TAR and potentially other locations and selectively transports HIV-1 RNAs, facilitating the unspliced RNA's nuclear export while retaining singly spliced RNAs in the nucleus. This is a novel role for this HIV-1 dependency factor.